The present invention relates to materials and methods of activating CD4+ T cells with specificity for particular antigenic peptides, the use of activated T cells in vivo for the treatment of a variety of disease conditions, and compositions appropriate for these uses.
Though the T cell repertoire is largely shaped during T cell development in the thymus, mature CD4+ T cells are also regulated extrathymically. Whereas, some conditions of activation lead to tolerance reflecting either anergy or clonal elimination, other conditions lead to a change in the type of response observed. For CD4+ T cells, this change in functional phenotype is largely a change in the pattern of cytokines produced. Although CD4+ T cells that are subject to acute activation maintain the ability to produce multiple cytokines, T cells obtained under conditions of chronic stimulation frequently demonstrate a more restricted pattern of cytokine production. For example, T cell clones maintained by repeated stimulation in vitro have defined two major functional categories of CD4+ T cells referred to as Th1 and Th2 type cells. Th1 type cells produce primarily interleukin-2 (IL-2), interferon-xcex3 (IFN-xcex3) and tumor necrosis factor (TNF), all of which are referred to as inflammatory cytokines. In contrast, Th2 type cells typically produce IL-4, IL-5, and IL-10 and are important for antibody production and for regulating the responses of Th1 type cells.
Although such extreme segregation in cytokine production is often not seen during in vivo T cell responses, recovery from certain types of infections, such as Leishmania, is associated with preferential production of IL-2/IFN-xcex3. Mice that mount a Th2 response to Leishmania fail to contain the infection and ultimately die. Inappropriate production of cytokines of the Th2 type response has been frequently linked to allergic type diseases such as asthma and contact sensitivity. For review on activation of CD4+ T cells and role in allergic disease, see Hetzel and Lamb, Clinical Immunol. Immunopath., 73:1-10 (1994).
Perhaps the strongest association of human disease with skewed patterns of cytokine production is the association of Th1 responses and Th1 type cytokines with autoimmune disease. Strong evidence in experimental models indicates that many types of autoimmunity including diabetes, experimental models for multiple sclerosis, autoimmune thyroiditis, and the like are mediated by Th1 type CD4+ T cells. The expression of Th2-associated cytokines, such as IL-4, in these models interfere with the development of autoimmune disease. Th2 type cytokines dampen the response of Th1 type cells while the Th1 type cytokines antagonize the development of Th2 type responses.
In view of the association of particular activated T cell subsets with particular disease conditions, a need therefore exists to be able to direct the proliferation and activation of CD4+ T cells to a desired T cells subset, a process that is extremely beneficial in altering the course of disease. One potential solution is to activate in vitro CD4+ T cells that are first isolated from a subject who may optionally be having either allergy or autoimmune conditions to produce cells secreting a preferred cytokine profile. The resultant activated T cells are then reintroduced to the subject to alter the course of disease and perhaps even provide a long term cure.
The challenge in this approach, now solved by the present invention, is the difficulty in defining activation conditions that reproducibly generate CD4+ T cell subsets that produce the desired therapeutic cytokine profile. Expression of particular cytokines is linked to a particular antigen presenting cell (APC) and their associated accessory (assisting) molecules. For a review of the surface proteins serving as accessory molecules that are involved in T cell costimulation, see Mondino and Jenkins, J. Leukocyte Biol., 55:805-815 (1994). Since both the cytokines produced by the APC and the coordinately expressed accessory molecules are themselves regulated by multiple factors, including the type of antigen, the affinity of the T cell receptor (TCR)-antigen interaction, antigen concentration and the like, predicting the outcome of T cell activation upon antigen presentation is historically very difficult. Indeed, as additional accessory molecules have been proposed for the activation process in vivo, it has become increasingly clear that many diverse molecules are involved in the regulation of T cell responses and act in combinatorial fashion to effect the outcome of T cell activation.
Prior to the present invention, the co-expression of selected MHC class II molecules in conjunction with one or more selected accessory molecules has not been possible. The present invention now presents a solution to predictably generate a preferred T cell phenotype through the reproducible activation of T cells to generate either Th1 or Th2 type T cells. The invention describes the generation of synthetic APC that present, in a neutral background, MHC class II molecules in combination with defined accessory molecules. The MHC class II molecules and defined accessory molecules are expressed in a nonmammalian insect cell and can be presented in a variety of forms of synthetic APC including insect cells displaying the molecules.
The advantage of using the insect cells as the expression and presentation vehicles for the MHC class II/accessory molecule compositions of this invention is that the cells do not endogenously produce regulatory cytokines and do not express mammalian accessory molecules. This overcomes the inherent unpredictability of using mammalian APC that express many molecules that are capable of altering the T cell response. In addition, the insect cell expression system described in the present invention provides for the expression of MHC class II molecules without bound peptide (i.e., xe2x80x9cemptyxe2x80x9d molecules) that can be produced under certain restrictive circumstances, such as temperature requirements. At physiological temperatures, these xe2x80x9cemptyxe2x80x9d molecules are normally unable to reach the cell surface as class II without bound peptide are very thermolabile. The invention utilizes the capacity of xe2x80x9cemptyxe2x80x9d MHC class II compositions to allow for the exogenous loading of selected peptides along with the ability to provided endogenously loaded counterparts.
A recombinant glycosyl-phosphatidylinositol (GPI)-modified MHC class I molecule (HLA-A2.1:GPI/xcex22m) was generated in the above-described insect cell system to produce antigen presenting cells as described in International Publication Number WO 96/12009 by Tykocinski. In that publication, the recombinant GPI-modified MHC class I molecules are isolated from the insect cell by affinity purification for subsequent reincorporation into cell membranes. In other aspects, the publication describes the preparation of a GPI-modified MHC class I molecule co-anchored on a cell membrane with a GPI-modified B7.1 costimulatory molecule. Although the publication states that GPI-modified MHC class II molecules can be prepared as described for those of MHC class I, the publication does not present any details for such preparation.
In contrast, the present invention provides and describes a unique means based on the co-expression of a selected MHC class II haplotype in conjunction with one or more accessory molecules, such as B7.1, to activate CD4+ T cells resulting in the differentiation to a particular T cell subset, Th1 or Th2 cells, that effect a preferred cytokine profile influence. The invention provides the advantage of selectively activating CD4+ T cells in vitro to a preferred T cell subset thereafter allowing for the reintroduction of the activated T cells into the patient. The present invention thus provides the ability to combine individual presenting molecules with particular accessory molecules for expression in selected combinations that permits reproducibility and predictability for selectively activating CD4+ T cells to a desired T cell subset not available in other approaches.
It has now been discovered that recombinant MHC class II molecules expressed in combination with selected accessory molecules, including costimulatory molecules and adhesion molecules, are effective in activating CD4+ T cells to become armed effector T cells that recognize target cells on which MHC class II heterodimer is expressed for complexation with peptide. Activation is characterized by proliferation and differentiation into effector T cell subsets, Th1 and Th2, that secrete particular cytokines. Th1 and Th2 type T cells are respectively referred to as inflammatory cells and T-helper cells.
Thus, the present invention relates to a synthetic antigen presenting system, also referred to as APC, for producing and presenting a mammalian, preferably human, MHC class II molecule in combination with one or more accessory molecules to activate CD4+ T cells.
In one embodiment, the system relates to a synthetic antigen presenting matrix having a support and at least the extracellular portion of a MHC class II heterodimeric molecule operably linked to the support and capable of binding to a selected peptide. The matrix also includes an accessory molecule operably linked to the support. The accessory molecule interacts with a specific receptor present on the CD4+ T cell. The MHC class II and accessory molecules are present in sufficient numbers to activate a population of CD4+ T cells specific for the MHC class II/peptide combination when the peptide is bound to the extracellular portion of the MHC molecule.
It has been found that an antigen presenting matrix having both a MHC class II heterodimer or at least the extracellular portion thereof loaded with a peptide specific for that MHC, together with an accessory molecule, provides a synergistic reaction in activating CD4+ T cells. Examples of accessory molecules are costimulatory molecules, including B7.1 and B7.2, adhesion molecules such as intercellular cell adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3), and survival molecules such as Fas ligand (FasL) and CD70. In some embodiments, the extracellular portion of such accessory molecules can also be used in the present invention.
The support used for the matrix can take several different forms. Examples for the support include solid support such as metals or plastics, porous materials such as resin or modified cellulose columns, microbeads, microtiter plates, red blood cells and liposomes.
Another type of support is a cell fragment, such as a cell membrane fragment. An entire cell is also contemplated as a support. In this embodiment, the matrix is actually cells which have been transformed with one or more expression vectors containing genes for the expression of MHC class II xcex1- and xcex2-chains along with at least one accessory molecule. The expressed proteins are then transported to the cell membrane where the transmembrane domain of the class II chains provide anchors allowing the extracellular domain to be displayed on the outer cell surface, thereby creating a synthetic antigen presenting cell (APC). The expression vectors contain the selected genes, preferably in the form of a cDNA sequence, operably linked to a promoter that is either constitutive or inducible.
The MHC xcex1- and xcex2-chains associate together forming a MHC class II heterodimer which binds to a peptide specific for that heterodimer. With the present invention, two methods of loading peptides onto MHC class II heterodimers are contemplated. In one embodiment, the peptide is loaded intracellularly following proteolytic processing of internalized intact protein into peptide fragments. The peptides are then loaded onto newly generated MHC class II molecules while they are still within the cell. Alternatively, the MHC class II molecules are expressed as empty molecules on the cell surface and synthetic or processed peptide reagents are then loaded extracellularly onto the MHC class II heterodimer.
Nucleotide sequences for encoding at least one accessory molecule gene operably linked to a promoter in a vector are also introduced into the cell. Following expression, the accessory molecule is coordinately anchored on the surface of the cell along with the MHC class II heterodimer in sufficient numbers to activate a population of CD4+ T cells lymphocytes specific for the MHC class II/peptide complex. Other molecules referred to as antigen processing assisting molecules are also contemplated for use in generating recombinant APC. These molecules are either provided by the cell used as APC or exogenously through an expression vector system as described above. Examples of such antigen processing assisting molecules include invariant chain, lysosomal enzymes and H2-M and H2-O molecules.
The cell line is synthetic in that at least one of the genes described above is not naturally present in the cells from which the cell line is derived. It is preferable to use a poikilotherm cell line because MHC molecules are thermolabile. A range of species are useful for this purpose. See, for example, U.S. Pat. No. 5,314,813 to Petersen et al. which discusses numerous species for this use, the disclosure of which is hereby incorporated by reference. Eukaryotic cells and preferably insect cells are used as APC. Preferred insect cells include Drosophila (fruit fly) and Spodoptera (butterfly).
MHC class II molecules have been expressed in insect cells such as Drosophila and Spodoptera cells. Since these cells do not have all the components of a mammalian immune system, the various proteins involved in the peptide loading machinery are absent from such cells. The lack of mammalian peptide-loading machinery allows the introduced mammalian MHC class II molecules to be expressed as empty molecules at the cell surface when the cells are cultured at thermostabile temperature restrictive conditions, such as at 28xc2x0 C. In contrast, at 37xc2x0 C., empty Class I molecules are thermolabile and tend to disintegrate. Thus, by incubating MHC class II-expressing Drosophila cells with peptides that specifically bind to anchored MHC class II molecule, virtually every class II molecule is loaded with one and the same peptide. Moreover, the invention provides for the means to introduce any known MHC class II xcex1- and xcex2-chain genes into an expression vector thereby overcoming the inherent limit to the number of MHC class II molecules expressed in any one mammal.
In the present invention, a specifically effective synergistic reaction in driving CD4+ T cells to a Th1-type response characterized by an increase in the cytokine interleukin-2 (IL-2) results from a Drosophila antigen presenting cell having MHC class II molecules bound with a peptide, a costimulatory molecule, and an adhesion molecule. In particular, a highly effective synergistic generation of IL-2 production coupled with CD4+ proliferation results from the combination of B7.2 and ICAM-1. In contrast, without ICAM-1 but with either B7.1 or B7.2, the Drosophila APC system loaded with peptide induced a Th2-type response characterized by an increase in IL-4. Thus, ICAM-1 antagonized the Th2-type response resulting in a Th1-type phenotype.
A Th1 phenotype characterized by IL-2 production coupled with proliferative responses also resulted from a synthetic antigen presenting cell having CD70 expressed simultaneously with ICAM-1 with or without B7.2.
Therefore, the selection of MHC class II genes in combination with at least one accessory molecule genes for expression thereof in an APC of this invention can be tailored depending upon the desired outcome for effecting proliferation and phenotypic activation of CD4+ T cells.
The present invention also relates to methods for making the synthetic APC systems as described above in which at least one expression vector containing genes for a MHC class II heterodimer and an accessory molecule is introduced.
Methods of producing activated CD4+ T cells in vitro are also contemplated. One preferred method comprises contacting, in vitro, CD4+ cells with a synthetic MHC class II/accessory molecule-bearing APC described above for a time period sufficient to activate, in an antigen-specific manner, a population of CD4+ T cells. The method may further comprise (1) separating the activated CD4+ cells from the antigen-presenting matrix; (2) suspending the activated CD4+ cells in an acceptable carrier or excipient; and (3) administering the suspension to an individual in need of treatment. As previously discussed, the antigens may comprise native or undegraded proteins or polypeptides, or they may comprise antigenic polypeptides which have been cleaved or synthesized into peptide fragments comprising at least 8 amino acid residues prior to incubation with the mammalian MHC class II heterodimeric molecules.
In addition to the utility of being able to direct the activation of CD4+ T cells to a desired T cell subset as described above, the ability to express any MHC class II molecule provides the means to identify unknown CD4+-activating peptide specific for that particular MHC class II molecule. As such, the present invention contemplates the activation of CD4+ T cells through the screening of a peptide library with synthetic APC expressing a particular MHC class II heterodimer.
In a further embodiment, the synthetic APC system described herein is useful for isolation of reactive CD4+ T cells from a heterologous population of cells. Such isolation provides the ability to monitor ongoing CD4+ T cell-mediated responses in disease conditions in a patient.
In another variation of the above, in view of the ability to selectively activate CD4+ T cells into a particular T cell subset for producing a preferred cytokine profile, the invention relates to methods of treating conditions in patients mediated by a undesirable CD4+ response. Such disease conditions characterized by either a Th1- or Th2-type response include autoimmune diseases, allergy and cancer. The therapeutic goal is to introduce CD4+ T cells activated to a preferred T cell subset to antagonize an ongoing CD4+ T cell response. Thus, the method comprises (1) obtaining a fluid sample containing resting or naive CD4+ cells from the patient; (2) contacting, in vitro, the CD4+ cells with a selected synthetic peptide-loaded APC of this invention for a time period sufficient to activate, in an antigen-specific manner, the CD4+ cells; and (3) administering the activated CD4+ cells to the patient.
Other embodiments are apparent to one skilled in the art.